Low-burst polymers and methods to produce polymers

ABSTRACT

A PLG copolymer material, termed a PLG(p) copolymer material, adapted for use in a controlled release formulation for a bioactive material is provided, wherein the formulation exhibits a reduced “initial burst” effect when introduced into the tissue of a patient in need thereof. A method of preparation of the PLG copolymer material is also provided, as are methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/527,377, filed Jun. 10, 2010, which is a nationalization under 35U.S.C. 371 of PCT/US2008/001887, filed Feb. 13, 2008 and published as WO2008/100532 A1 on Aug. 21, 2008, which claims the priority ofprovisional application U.S. Ser. No. 60/901,435, filed Feb. 15, 2007,which applications and publication are incorporated herein by referenceand made a part hereof.

FIELD OF THE INVENTION

The field of the invention is improved lactide/glycolide copolymers forcontrolled release in vivo of bioactive substances, wherein the initialburst effect is reduced.

BACKGROUND

Compositions adapted for use in controlled release delivery systems,such as biodegradable and bioerodible implants, are known. See, forexample, U.S. Pat. Nos. 7,019,106; 6,565,874; 6,528,080; RE37,950;6,461,631; 6,395,293; 6,355,657; 6,261,583; 6,143,314; 5,990,194;5,945,115; 5,792,469; 5,780,044; 5,759,563; 5,744,153; 5,739,176;5,736,152; 5,733,950; 5,702,716; 5,681,873; 5,599,552; 5,487,897;5,340,849; 5,324,519; 5,278,202; and 5,278,201. Such controlled releasesystems are in general advantageous as they provide for the controlledand sustained release of medications, often directly at or near thedesired site of action, over the period of days, weeks or even months.Controlled release systems include polymer matrices that are known to bebroken down in the body by various endogenous substances such as enzymesand water, such as polyesters including poly-lactide, poly-glycolide,and copolymers thereof (“PLG copolymers”) prepared from glycolide(1,4-dioxan-2,5-dione, glycolic acid cyclic dimer lactone) and lactide(3,6-dimethyl-1,4-dioxan-2,5-dione, lactic acid cyclic dimer lactone).These copolymer materials are particularly favored for this applicationdue to their facile breakdown in vivo by water or enzymes in the body tonon-toxic materials, and their favorable properties in temporallycontrolling the release of biologically active agents (“bioactiveagents”) that may be contained within a mass of the polymer.

The release of many bioactive agents such as peptides, proteins, andsmall molecule drugs from controlled release systems can occur at ahigher than optimal rate during the first 24 hours after implantationunder certain conditions. This is known in the art as the “burst effect”or the “initial burst effect,” and is potentially undesirable, asoverdosing can result.

U.S. Pat. No. 4,728,721 discusses the presence of water-solubleunreacted monomers and water-soluble low molecular weight oligomerswithin the copolymers that are used to form microcapsules into whichbioactive agents are incorporated. According to the inventors therein,the presence of these impurities tends to increase the initial bursteffect, although the mechanism by which this burst occurs is undefined.The patent provides methods for removal of some of these impurities bywashing of a solid form of the polymer with water, or by dissolving thepolymer in a water-soluble organic solvent and adding the solution towater. The patent states that the ratio between the water and thepolymer being purified is not critical, but that water should be used inlarge excess. Removal is effected exclusively of water-soluble materialssuch as lactic acid, glycolic acid, and very low molecular weightoligomers by this method.

U.S. Pa. No. 5,585,460 discusses the processing of polymers used for thepreparation of microcapsules, wherein polymers produced without use of acatalyst are dissolved in a water-soluble organic solvent andprecipitated in water, to provide polymers that are stated to havecomponents with molecular weights under 1,000 (1 kD) of less than about3%. In the '460 patent, the inventors therein state that the processclaimed in the U.S. Pat. No. 4,728,721 patent, discussed above, producesa polymer that, while it does reduce the amount of the initial release,also reduces the rate of release in later stages, whereas the method ofthe '460 patent allows for suppression of the initial burst whileproviding an increased rate of release at a later point in time.

U.S. Pat. No. 4,810,775 describes a process for purifying partlycrystalline or amorphous polymers wherein high shear forces are appliedat the time of contacting the polymer with a precipitating agent such aswater so that minute particles of the polymer are obtained. This patentdescribes that such treatment results in the removal of residualmonomers and catalysts from the polymer.

U.S. Pat. No. 7,019,106 discusses a process for producing a lactic acidpolymer of 15,000 to 50,000 in weight-average molecular weight, thecontent of polymeric materials having not more than about 5,000 inweight-average molecular weight therein being not more than about 5% byweight. The process is characterized by hydrolysis of a high molecularweight lactic acid polymer and precipitation of the hydrolyzed product,which is stated to provide for a reduced burst effect. Desirablesustained release properties are attributed in part to a relatively highacid content per gram of copolymer.

However, despite these attempts to reduce the burst effect in controlledrelease compositions, there remains a need for compositions wherein theinitial burst effect is reduced or minimized. This need is especiallystrong in the field of flowable compositions and injectable masses ofcontrolled release compositions, as opposed to microcapsules, whereinphysically larger masses of the polymer than are found in microcapsulesare implanted in body tissue to provide for sustainable controlledrelease over longer periods of time.

SUMMARY OF THE INVENTION

The copolymers of the present invention when used in, for example, thecontrolled delivery systems known as liquid delivery systems, otherwiseknown as flowable delivery systems, like the Atrigel® systems that aredisclosed in U.S. Pat. Nos. 6,565,874, 6,528,080, 6,461,631, 6,395,293,and references found therein, provide for substantially improved releaserates for a bioactive agent, both a reduced initial burst and adesirable long-term sustained rate of release.

Unexpectedly, it has been discovered that use of these copolymermaterials in the flowable delivery system effectively reduces theinitial burst effect in the release of bioactive agents from thecontrolled release formulation after its implantation within livingtissue, without loss of desirable long-term sustained rates of releaseof bioactive agents, particularly for those systems adapted to release abioactive agent over a relatively prolonged period, such as 30 days to 6months.

The present invention provides a biocompatible, biodegradable PLGlow-burst copolymer material, termed a PLGp or a PLG(p) copolymer,adapted for use in a controlled release formulation, the low-burstcopolymer material being characterized by a weight average molecularweight of about 10 kilodaltons to about 50 kilodaltons and apolydispersity index of about 1.4-2.0, and being further characterizedby having separated therefrom a copolymer fraction characterized by aweight average molecular weight of about 4 kDa to about 10 kDa and apolydispersity index of about 1.4 to 2.5 (hereinafter the “removedcopolymer fraction”). The inventive PLG low-burst copolymer material isprepared from a starting PLG copolymer material without a step ofhydrolysis of a higher molecular weight PLG copolymer material, bydissolving the starting copolymer material, which is not a product ofhydrolysis of a higher molecular weight PLG copolymer material, in asolvent, then precipitating the inventive low-burst copolymer materialwith a non-solvent. This process, as applied to a starting material thathas never been subjected to hydrolysis, separates out an amount of theremoved copolymer fraction effective to confer desirable controlledrelease properties including low initial burst upon the copolymer of theinvention.

The starting PLG copolymer material can be prepared by any suitablemeans, including ring-opening polymerization of cyclic dimeric esterslactide and glycolide and condensation of lactic and glycolic acids.Preferably, the ring-opening polymerization of lactide and glycolide isused to prepare the starting copolymer from which the low-burst PLGcopolymer of the invention is prepared. The ring-opening polymerizationreaction, which can be a catalyzed reaction, for example using a tinsalt such as stannous octanoate as a catalyst, incorporates two lactateor two glycolate units at a time as the polymerization progresses.

It is well known that a weight average molecular weight of a polymermaterial or fraction of a polymer material describes an average propertyderived from the individual molecular weights of all the individualpolymer molecules making up the material or fraction. For any givenweight average molecular weight that a polymer material or fraction mayhave there are many possible distributions of individual molecularweights of the molecules making up the material or fraction. Thus, inthe present invention, the removed copolymer fraction having a weightaverage molecular weight of about 4 kDa to 10 kDa can include copolymermolecules with individual molecular weights ranging from a few hundred(oligomers) up to well in excess of 10 kDa. There are many differentcombinations of individual molecular weights that can yield any givenvalue of the weight average molecular weight of a polymer sample. Thebreadth of the distribution of the individual molecular weights of thecopolymer molecules making up the removed copolymer fraction of theinvention is at least partially expressed by the polydispersity index,which can range from about 1.4 to about 2.5. Whatever the distributionof individual molecular weights may be in the removed copolymerfraction, the mass of the removed copolymer fraction amounts to about2-20% of the sum of the masses of the removed copolymer fraction and thePLG low-burst copolymer material obtained thereby, more preferably about3-15% of the sum of the masses, and yet more preferably about 5-10% ofthe sum of the masses. Typically, the greater the weight averagemolecular weight of the removed copolymer fraction within the definedrange of about 4 kDa to 10 kDa, the greater is the weight averagemolecular weight of the inventive PLG low-burst copolymer materialwithin the range of about 10 kDa to about 50 kDa.

The present invention provides a PLG low-burst copolymer materialcomposed of a set of individual PLG copolymer molecular chains. Apredominant proportion of these molecular chains predominantly includelactide/lactate residues adjacent to at least one end of each copolymermolecular chain and predominantly include glycolide/glycolate resides ininternal domains of each copolymer molecular chain. It is believed thatthis distribution of lactide/lactate versus glycolide/glycolate units inthe inventive copolymers may be responsible for their unexpected lowburst and desirable sustained release properties.

The present invention further provides a method of preparation of a PLGlow-burst copolymer material, wherein a removed copolymer material isseparated from a starting PLG copolymer material by a step of dissolvingthe starting copolymer material in a solvent and precipitating thelow-burst copolymer material by admixture of a non-solvent, without anystep of hydrolysis of a higher molecular weight PLG copolymer being usedin the process. The method of the present invention requires avoidanceof a step of hydrolysis of a higher molecular weight copolymer materialin order to provide a low-burst copolymer material of the invention. Theinventive low-burst copolymer material exhibits surprisingly low initialburst properties as well as a surprisingly high sustained releaseeffect. It is believed that this unexpectedly favorable low-burstproperty arises from the differing distributions of the more lipophiliclactate/lactide units adjacent to at least one end of the polymer chainsin the present inventive polymer versus a polymer prepared with a stepof hydrolysis. Copolymers prepared by a method including a step ofhydrolysis can have a greater predominance of polymer chains that havethe less lipophilic glycolate or glycolide units adjacent to both themolecular chain ends due to the hydrolysis of ester bonds inglycolate/glycolide rich internal domains.

In a low-burst PLG copolymer material prepared from a starting PLGcopolymer that was made without using a core initiator, i.e., a PLGcopolymer having a carboxyl group at one end of each chain and ahydroxyl group at the other end, the acid content per gram is lower inan inventive polymer than in a PLG copolymer prepared by a methodincluding a step of hydrolysis of a higher molecular weight polymer, butthe low-burst property of the inventive polymer is surprisingly at leastas good as or better than that of the polymer prepared with a step ofhydrolysis.

The relatively low acid content of the low-burst copolymers of theinvention can be advantageous because the inventive copolymer materialsuffers from less acid-catalyzed auto-hydrolysis over time. If thestarting PLG copolymer material comprises a PLGH, or acid terminatedcopolymer, the inventive process decreases the acid content per unitmass by removal of oligomers. The implication of a lower auto-hydrolysisrate of the polymer is that, for example, when implanted in the tissueof a patient, this lessening of auto-hydrolysis of the inventivecopolymer enables a smooth monotonic, long lasting release profile ofthe bio active agent contained in a controlled release formulation, thecopolymer also possessing a low initial burst.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a study of the percentage release ofoctreotide from inventive copolymers versus control copolymers as afunction of time.

FIG. 2 shows the degree of octreotide release from a controlled releaseformulation as a function of polymer type.

DETAILED DESCRIPTION OF THE INVENTION Definitions of the Invention

In the present application, the terms “burst effect” or “initial bursteffect” are used to refer to the burst effects in which a higher thanoptimal rate of diffusion of a bioactive agent out of a controlledrelease formulation occurs during the solidification of a liquiddelivery system and/or during the initial period following implantationof a preformed solid implant such as a monolithic or a microparticulateimplant. It is believed that the copolymers according to the presentinvention are particularly suitable for controlling this initial burst.

A “liquid delivery system” or a “flowable delivery system” is acombination of polymer, bioactive agent and organic solvent, such as inthe Atrigel® system. Upon injection of the flowable material intotissue, the solvent disperses into the tissue and body fluid diffusesinto the injected bolus, thereby causing coagulation of the polymer intoa solid or semi-solid mass. Often, dispersion of the solvent out of themass will carry the bioactive agent with it into surrounding tissues,thereby producing a burst effect. Solvents that can be used with theinventive polymers for a liquid or flowable delivery system includeN-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide,dimethylsulfoxide, polyethylene glycol 200, polyethylene glycol 300, ormethoxypolyethylene glycol 350.

A solid implant, of the monolithic or of the microparticulate type, alsodisplays a burst effect due to the presence of bioactive agent on andnear the surface of the implant, and due to the presence of easilyleached bioactive agent within the micro-channels and mesopores thatform within the implant as a result of its initial interaction with bodyfluid.

The term “low-burst” as used herein, such as a “low-burst copolymermaterial,” refers to a phenomenon wherein this burst effect is minimizedor reduced relative to that observed from a comparable art copolymercomposition, while maintaining a desirable long-term release profile.

The terms “polymer” or “copolymer” as used herein refer to substantiallylinear polyesters, also referred to herein as “PLG copolymers,”predominantly formed of monomeric lactate and glycolate hydroxyacids, orlactide and glycolide dimeric hydroxyacids, and include compositionsreferred to in the art as poly(lactate-glycolate),poly(lactate(co)glycolate), poly(lactide-glycolide), poly(lactide(co)glycolide), PLG, PLGH, and the like, with the understanding thatadditional moieties may be included, such as core/initiator groups (forexample, diols, hydroxyacids, and the like), capping groups (forexample, esters of terminal carboxyl groups, and the like) and otherpendant groups or chain extension groups covalently linked to or withina polyester backbone, including groups that cross-link the substantiallylinear polyester molecular chains, without departing from the meaningassigned herein. PLG copolymers, as the term is used herein, includesmolecular chains with terminal hydroxyl groups, terminal carboxyl groups(i.e., acid-terminated, sometimes termed PLGH) and terminal ester groups(i.e., capped).

As used herein, the term “polymer material” or “copolymer material”refers to the physical assembly or the combined mass of a plurality ofindividual polymer or copolymer molecules (molecular chains) in a givensample, respectively, each of which molecules (molecular chains) has itsown defined molecular weight in the usual chemical sense of the word. A“polymer material” or “copolymer material” as used herein usually iscomposed of a set of individual polymer or copolymer molecules havingvarious different individual molecular weights. Thus, when the molecularweight of such a polymer material or a copolymer material is referredto, it is an average molecular weight. Without further characterization,such an average molecular weight is a weight average molecular weight asused herein. The full description, weight average molecular weight, maybe used synonymously. If the average molecular weight being referred tois the number-average molecular weight, it will be explicitly stated inthis specification. When the individual molecular weights of thecomponent individual molecules (molecular chains) is referred to, theterm “individual molecular weight” is used in this specification. Weightaverage molecular weights are determined by the use of gel permeationchromatography (GPC) with reference to polystyrene standards, as is wellknown in the art.

The term “polydispersity index” as used herein is defined as theweight-average molecular weight of a sample of a polymer materialdivided by the number-average molecular weight of the sample of thepolymer material. The definitions of the terms “weight-average molecularweight” and “number-average molecular weight” are well-known to those ofskill in the art. The polydispersity index is well-known to characterizethe distribution of molecular weights in a polymer. The higher the valueof the polydispersity index, the broader the spread of individualmolecular weights of the polymer molecular chains making up the polymermaterial. The lower the value of the polydispersity index, the moreuniform and tightly grouped are the individual molecular weights of theindividual polymer molecules making up the polymer material in question.In the unlikely event that every polymer molecule in the polymermaterial were identical, the weight-average molecular weight and thenumber-average molecular weight would be identical, and thus thepolydispersity index (“PDI”) would be unity.

The terms “lactate” and “glycolate” as used herein, depending uponcontext, refer to either the hydroxyacids, lactic acid and glycolic acidrespectively, or their salts (lactates and glycolates) which are used asreagents in preparation of inventive copolymers, or refer to thosemoieties as residues incorporated via ester bonds into the inventivepolyester molecular chains. When a copolymer is formed by polymerizationof lactic acid (lactate) and glycolic acid (glycolate), each molecularchain consists of individual lactate and glycolate monomeric unitsincorporated into the copolymer molecular chain. The terms “lactide” and“glycolide” as used herein, depending upon context, refer to either thecyclic dimeric esters of lactate and glycolate respectively whenreferring to reagents used in preparation of inventive copolymers, orrefer to those segments as incorporated ring-opened dimers in the formedpolymer molecular chains. Thus, a statement about polymerization oflactide and glycolide refers to a polymerization reaction of the cyclicdimeric esters, whereas a statement about a lactide or glycolide residuewithin a copolymer molecular chain refers to that grouping of atoms,ring-opened, and incorporated into the copolymer chain. When a copolymeris formed by polymerization of lactide and glycolide, each incorporatedlactide or glycolide residue is believed to consist of a pair of lactateor glycolate monomeric units, respectively. It is understood that when alactide and glycolide residue in a copolymer molecular chain is referredto, the terms mean double (dimeric) units of two lactate (L-L), or twoglycolate (G-G), residues in the molecular chain, respectively, such asis believed to result from the polymerization of lactide and glycolide.

When a lactate (L) or a glycolate (G) residue in a copolymer molecularchain is referred to, the terms mean single lactate (L) or glycolate (G)residues in the molecular chain, respectively, which can be within alactide (L-L) or a glycolide (G-G) residue if the given lactate orglycolate is adjacent to another lactate or glycolate residue,respectively, regardless of the method used to prepare the copolymermolecular chain. As is most polymeric systems, this arrangement ofresidues is not all or none. Instead, the arrangement is a predominance.Thus, for the lactide and glycolide copolymers, a predominance of L-Land G-G residues will be present with some L and G (single) residuesalso present. The chemical reason underlying this characterization isthe polymerization process. During polymerization, growing polymerchains are broken and reformed. This scission may split dimer residuesand recombine single residues. For the lactate and glycolate copolymers,a predominance of L and G (single) residues will be present. This kindof polymer will have a relatively few sequences including repeats ofdimer residues because of entropy factors.

It is understood that when the terms “lactic acid,” “lactate,” or“lactide” are used herein, that any and all chiral forms of thecompounds are included within the terms. Thus, “lactic acid” includesD-lactic acid, L-lactic acid, DL-lactic acid, or any combinationthereof; “lactide” includes DD-lactide, DL-lactide, LD-lactide,LL-lactide, or any combination thereof.

A substantially linear molecular chain as is formed by a polymerizationprocess, such as a copolymer molecule that is within a copolymermaterial of the invention, has two ends, each end with a nearby “enddomain,” and an “internal domain” between the end domains. The terms arenot exact, but rather describe general regions of a copolymer molecularchain, each end domain being the approximately 10-20% of the totallength of the chain terminating at each of the two chain ends, and theinternal domain being the remaining approximately 60-80% of the chainthat lies between the end domains.

A “solvent” is a liquid, usually organic, that serves to dissolve acopolymer material to provide a homogeneous solution of the copolymermaterial. The term “non-solvent” refers to a precipitation solvent, thatis a usually organic liquid, that is not a solvent for the copolymer. Itis in this context that the term “non-solvent” is used herein. Twoliquids, such as a solvent and a non-solvent, are “miscible” when theycombine with each other in all proportions without phase separation.Solvents may be “soluble” in each other but not “miscible” when they cancombine without phase separation in some, but not in all, relativeproportions.

The preparation of an inventive low-burst copolymer material is carriedout “without a step of hydrolysis of a higher molecular weight PLGcopolymer material.” By this is meant that, following the initialcopolymerization of the monomers lactate and glycolate, or lactide andglycolide, to prepare a starting material for preparation of theinventive low-burst copolymer material, no conditions are applied, suchas treatment with acid or alkali, that would hydrolyze ester bondsbetween adjacent monomeric units in the polymer. Therefore, a “highermolecular weight PLG copolymer material” as the term is used hereinrefers to a PLG copolymer material of a weight-average molecular weightthat is greater than the weight average molecular weight possessed by acombination of the PLG low-burst copolymer material of the inventionplus the removed copolymer fraction, such as exists in the starting PLGcopolymer material prior to the step of separation of the removedcopolymer fraction from the PLG low-burst copolymer material. This kindof hydrolysis does not refer to complete hydrolysis of a PLG copolymerback to its constituent monomers (lactate and glycolate), but rather toa step of partial hydrolysis whereby longer molecular chains are cleavedto yield shorter molecular chains, as is the case with certain artpolymers adapted for use in controlled release formulations. Therefore,following the polymerization reaction, of whatever type it may be, thatprovides the starting PLG copolymer material, no step of hydrolysis isinterposed prior to the separation of the removed copolymer fractionfrom the PLG low-burst copolymer material in the method of theinvention, and the product of the invention has therefore not beensubjected to a hydrolysis step. As discussed below, this absence ofhydrolysis has implications for the distributions of lactide/lactateversus glycolide/glycolate units at the end domains of and in theinternal domains of the molecular chains making up the inventive PLGlow-burst copolymer material. As discussed above, PLG copolymer chainsare enriched in G residues near the site of initiation of thepolymerization reaction, and enriched in L residues in the regionsincorporated late in the polymerization reaction. This implies that inPLG copolymer materials synthesized using, for example, a diol core fromwhich polymerization proceeds in both directions, the internal domainsof the polymer molecule near the core will be G-rich and both ends willbe L-rich. In contrast, a PLG copolymer material of the PLGH type, whichis polymerized from a lactic acid initiator, wherein polymerizationtakes place only at the hydroxyl end of the lactic acid, will be G-richat the end of the molecular chain adjacent to the initiating lactic acidand L-rich at the distal end of the chain that is formed late in thepolymerization reaction.

The term “acid content per unit mass” when used herein refers to thecontent of carboxylic acids, which are titratable using standardprocedures well known in the art, divided by a unit mass such as 1 gram.PLG copolymers, being chains of hydroxyacids joined by ester bonds,typically have a single titratable carboxylic acid group at one end ofthe molecular chain. Thus, a sample of a copolymer made up of shortmolecular chains has a higher acid content per unit mass relative to asample of a copolymer made up, on average, of longer (higher molecularweight) molecular chains. The sample made up of shorter, lower molecularweight chains has relatively more individual polymer chains and thusrelatively more carboxylic acid groups per gram.

The inventive copolymer material is also known as a “PLGp” or a “PLG(p)”copolymer, the subscript “p” referring to “purified.”

DETAILED DESCRIPTION OF THE INVENTION

The low-burst copolymer materials of the present invention areparticularly useful in reducing the initial burst effect in controlledrelease formulations such as those of the Atrigel® type. The inventivecopolymer material (“low-burst copolymer material”) is characterized asbeing a derived from a sample of a PLG starting copolymer (“startingcopolymer material”). The low-burst copolymer material is preparedwithout the use of a step of hydrolysis of a high molecular weight PLGcopolymer. The inventive low-burst copolymer material is characterizedby a weight average molecular weight of about 10 kilodaltons (kDa) toabout 50 kDa and a polydispersity index of about 1.4-2.0. The low-burstcopolymer material is obtained from a starting PLG copolymer materialthat is prepared by any suitable polymerization method but not includinga step of hydrolysis in its preparation, from which a copolymer fraction(“removed copolymer fraction”) that is characterized by a weight averagemolecular weight of about 4 kDa to about 10 kDa and a polydispersityindex of about 1.4 to 2.5, has been removed.

The inventive copolymer material from which the removed copolymerfraction has been separated is prepared by purification from a PLGstarting copolymer material. The PLG starting copolymer material is nota reaction product resulting from hydrolysis of a high molecular weightpolymer, but otherwise can be made according to any of the standardmethods well-known in the art, such as condensation polymerization of amixture of lactate and glycolate, or ring-opening polymerization of amixture of lactide and glycolide. Preferably, the ring-openingpolymerization of lactide and glycolide is used to prepare the startingcopolymer from which the low-burst PLG copolymer of the invention isprepared. The ring-opening polymerization reaction, which can be acatalyzed reaction, for example using a tin salt such as stannousoctanoate as a catalyst, incorporates two lactate or two glycolate unitsat a time as the polymerization progresses

In the inventive process, the removed copolymer fraction is separatedfrom the starting copolymer material by dissolving the startingcopolymer material in a solvent, then by adding a non-solvent toprecipitate the low-burst polymer, and then collecting the inventivelow-burst copolymer material, leaving the removed copolymer fraction inthe supernatant.

The separation of the removed copolymer fraction that is characterizedby a weight average molecular weight of about 4 kD to about 10 kD and apolydispersity index of about 1.4 to 2.5, to yield the low-burstcopolymer material may be accomplished by methods according to thepresent invention. The separation is carried out by dissolution of thestarting copolymer material in a solvent and precipitation of thelow-burst copolymer material by mixture of this solution with anon-solvent. The solvent and non-solvent can be miscible. Specifically,the polymer can be dissolved in dichloromethane and precipitated withmethanol.

In one embodiment according to the present invention, the low-burstcopolymer material can have a weight average molecular weight of about15 kDa to about 50 kDa, and a polydispersity index of about 1.4-1.8.Compared to the starting copolymer material from which the removedcopolymer material has been separated, not only are the weight-averageand the number-average molecular weights of the low-burst copolymermaterial somewhat greater, but even more significantly, the width of thespread of the individual molecular weights of the copolymer molecules isless, i.e., the molecular weight distribution is narrower. Thisnarrowness is reflected in the relatively low polydispersity index ofthe low-burst copolymer according to the present invention.

When an inventive low-burst copolymer material was formulated as part ofa controlled release system, such as the Atrigel® system, it wassurprisingly found that a reduction of the initial burst effect in therelease of a variety of peptide or protein bioactive agents wasobserved. This reduction was demonstrated by measurement of the amountof bioactive agent released from the controlled release system as afunction of time. The low-burst copolymer material of the presentinvention, which is adapted to be used in the Atrigel® system, interalia, was compared to the same formulation containing a polymer that wasnot purified by the inventive method. The formulation containing thelow-burst copolymer material of the invention displayed a lower drugrelease in the first 24 hours and later time points. Thus, use of thelow-burst copolymer in the Atrigel® system demonstrates a simple,effective process to improve in vivo drug release kinetics, especiallywith respect to drug release during the first 24 hours afteradministration.

The starting copolymer material can be prepared by any means known inthe art, such as: polymerization of a mixture of the cyclic dimeresters, lactide and glycolide, for example with a catalyst such asstannous octanoate, with or without a core/initiator such as lactic acidor a diol; polymerization of a mixture of lactic acid and glycolic acid,for example with an acid catalyst, under dehydrating conditions; or anyother suitable method. The starting copolymer material is not subjectedto a step of hydrolysis prior to the steps of separation. Thisnon-hydrolysis factor is believed to be significant in providing theunexpected low-burst properties of the inventive copolymer materials.

It is well known in the art that in the polymerization of lactide andglycolide in the presence of a catalyst, a suitable means for preparingthe starting copolymer material of the invention, the glycolidemolecules react in the ring-opening polymerization reaction at a higherrate than do the lactide molecules, due to the lesser steric hinderanceof glycolide relative to lactide (lactic acid bearing a methyl group inplace of a hydrogen atom of glycolic acid). This results in theearly-polymerizing regions of the growing copolymer chain predominantlyderiving from glycolide incorporation. As the glycolide concentration inthe reaction mixture drops during the course of the polymerizationprocess due to this selective depletion of monomer, thelate-polymerizing regions of the copolymer chain predominantly arederived from lactide incorporation. Thus, as polymerization occurs inboth directions, the internal regions or internal domains of themolecular chains are composed predominantly of glycolide residues, andthe ends of the chains are composed predominantly of lactide residues.By “predominantly” is meant herein that the one component, lactide orglycolide, is found more frequently than the other component; i.e., apredominantly glycolide-incorporating or glycolide-containing domain orregion of a copolymer chain has more glycolide residues than lactideresidues in the domain on a molar basis as defined relative to the molarconcentrations of the monomers in the starting reaction mixture; or, inother words, glycolide is over-represented in that region or domain ofthe polymer relative to its initial proportion in the polymerizationreaction mixture. In a predominantly glycolide- or glycolate-containingdomain, glycolide/glycolate residues are found at a higher molarpercentage in that domain than they represent in the starting reactionmixture, and lactide/lactate residues are found at a lower molarpercentage in the domain than they represent in the starting reactionmixture.

The difference in distribution of lactide/lactate vs.glycolide/glycolate moieties along the polymer chain will vary fromslight to significant depending upon the reaction time allowed for postpolymerization rearrangement. This post-polymerization period isbalanced against increasing weight average molecular weight of thecopolymer material. Accordingly, within the weight average molecularweight parameters of this invention, the difference in distribution willbe moderate to significant, preferably in the range of 5 to 35%, morepreferably 10-25%, on a molar basis.

Thus, the molecular chains making up a low-burst copolymer material ofthe invention, as a result of the method of preparation either fromlactate/glycolate or from lactide/glycolide without a step of hydrolysisfollowing polymerization, are believed to have predominantlylactide/lactate residues in the end domains of the molecular chains andglycolide/glycolate residues in the internal domains of the molecularchains. It is well-known in the art that lactide/lactate residues have ahigher degree of hydrophobicity than do glycolide/glycolate residues, asa result of the presence in lactide/lactate residues of a hydrophobicmethyl group. Based on this fact, it is believed that a low-burstcopolymer material of the invention can present a more hydrophobicdomain to its surroundings, as the ends of the chains are likely moreaccessible to other molecules in the surrounding environment. Thisenhanced hydrophobicity of the chain end domains may be a cause of theunexpected low-burst properties of the inventive copolymers. While notwishing to be bound by theory, it is believed that this degree ofhydrophobicity may cause, at least in part, the unexpected but desirablelow-burst properties of an inventive polymer relative to art polymersdue to its hydrophobic interactions with the contained bioactive agentand resulting changes in the partition coefficients of the bioactiveagent between the copolymer matrix and the surrounding solutions of bodyfluids when implanted in a patient.

An art copolymer, such as can be prepared by hydrolysis of a highmolecular weight precursor copolymer, is believed to differ from aninventive polymer in that the molecular chains making up the artcopolymer material do not have predominantly lactide/lactate containingdomains at both ends of the molecular chains. This difference is theresult of hydrolysis of a high molecular weight precursor. Uponhydrolysis of a high molecular weight precursor polymer, the resultingcleavage causes one end (the newly formed end) to contain predominantlyglycolide/glycolate residues rather than lactide/lactate residues. Thiseffect occurs to a great extent within the interior domain on a purelystatistical basis, and is further enhanced by the well-known fact of thereduction of the rate of ester hydrolysis reactions due to sterichinderance. Thus, less hindered ester bonds (such as glycolate bonds asopposed to lactate bonds) are expected to hydrolyze at a higher rateunder given conditions than are more hindered ester bonds. As a result,hydrolysis of the ester bond between adjacent glycolate residues (G-G)is believed to take place more readily, at a higher rate, thanhydrolysis of the ester bond between a lactate and a glycolate residue(L-G or G-L) which is likewise believed to take place more readily, at ahigher rate, than hydrolysis of the ester bond between to lactateresidues (L-L). As a consequence, in a copolymer chain that consists ofall three types (G-G, G-L/L-G, and L-L) of ester bonds, an ester bondwould be more frequently cleaved at the G-G ester linkages than at anyof the other types of ester linkages, with L-L ester linkages occurringleast often at the lowest relative rate. Thus, a G-G rich domain such asthe internal domain of the copolymer will more frequently be the site ofhydrolysis than any other domain. Therefore, a copolymer molecular chainthat has undergone hydrolysis will yield, as a reaction productcopolymer, molecular chains that will tend to have at least one end ofthe product chain or possibly both ends of a product chain formedpredominantly of glycolide/glycolate residues, rather than being formedpredominantly of lactide/lactate residues as in the inventivecopolymers.

As a result, copolymer materials that have been prepared by a methodincluding a step of hydrolysis of a high molecular weight copolymerchain will be made up of copolymer molecular chains that have more endsformed predominantly of glycolide/glycolate residues than oflactide/lactate residues. This would be expected to result in a lesshydrophobic environment that the end regions of these copolymermolecular chains present to the surrounding environment, and may accountfor the less desirable high initial burst properties of art copolymersprepared by the hydrolysis method compared to the more desirable lowinitial burst properties of inventive copolymers as disclosed andclaimed herein.

As a consequence of the above-discussed rate of incorporation and rateof hydrolysis factors, the removed copolymer material of the presentinvention is also different than copolymer fractions that may be removedin art processes using solvent/non-solvent precipitation techniques. Theart copolymer for use in controlled-release formulations that has beenprepared by a method including hydrolysis of a high molecular weightcopolymer, following by dissolution in a solvent and precipitation of afraction of the hydrolyzed copolymer with a non-solvent, will not onlyhave different distributions of lactide/lactate (L) andglycolide/glycolate (G) in the precipitated fraction, but the artnon-precipitated material will also have different distributions of Land G along the molecular chains compared to the non-precipitatedfraction of the present invention. The non-precipitated, typically lowermolecular weight, copolymers resulting from a process involvinghydrolysis would likewise be expected to have a higher proportion of Gresidues at or near the chain termini than copolymers that had notundergone a hydrolysis step. Furthermore, due to the unexpectedly goodlow-burst properties of the inventive polymers, the acid content of acopolymer used in a controlled release formulation such as an Atrigel®system can be reduced yet still achieving a comparable decrease in theundesired burst effect. It is well known in the art that a higher acidcontent per unit mass can diminish the undesired burst effect, and artcopolymers used in this application have been tailored to achieve thisresult. However, from another perspective a relatively higher acidcontent per unit mass is undesirable, in that the rate of auto-catalyzedhydrolysis of the PLG copolymer ester bonds would be greater due to thehigher acid catalyst concentration in situ. Auto-hydrolysis of copolymerester bonds is known to result in more rapid decomposition of thepolymer, which would tend to interfere with achieving a desirablesmooth, monotonic release of the bioactive ingredient formulated withthe copolymer in a controlled release preparation such as Atrigel®.

Therefore, the inventive products by process can be clearlydistinguished structurally over the products produced by a step ofhydrolysis of high molecular weight copolymers.

The starting copolymer of the present invention can be prepared by anyavailable method, not including a step of hydrolysis of a high molecularweight copolymer, but including ring-opening polymerization of mixturesof lactide and glycolide precursors, dehydrative polymerization oflactic acid and glycolic acid, and the like. Purification of thestarting copolymer by a method of the invention is carried out bydissolving the starting copolymer material in a solvent, for example,dichloromethane or any other suitable organic liquid. Precipitation iscarried out by contacting that solution with a non-solvent, for exampleeither by adding the copolymer solution to a volume of a non-solvent, orby adding a volume of a non-solvent to the copolymer solution. Anexample of a typical non-solvent is methanol. Preferably, the solventand the non-solvent liquids are miscible, or at least substantiallysoluble, in each other. The mixing of the copolymer solution and thenon-solvent can take place under a wide variety of temperatures,concentrations, and modes of mixing.

A copolymer of the invention can be used to advantage in a number ofdiffering types of controlled release formulations, each of which canembody a variety of different bioactive agents and used for thetreatment of different malconditions. The low-burst property of theinventive polymers are particularly well-suited to use with bioactiveagents wherein overdose and potential toxicity of the agent are ofmedical concern, as well as with bioactive agents with which it ismedically indicated to maintain a relatively constant dosage over aprolonged period of time.

Examples of bioactive agents that can advantageously be used withcontrolled release formulations incorporating a copolymer of theinvention include leuprolide and related peptide analogs useful formodulating LHRH levels; steroids such as can be used for birth control,treatment of cancers such as breast cancer, and the like;prostaglandins, such as latanoprost and travoprost that can be used fortreatment of glaucoma; analgesics, such as oxycodone, for treatment ofchronic pain; carbonic anhydrase inhibitors such as brinzolamide anddorzolamide, useful for treatment of glaucoma and hypertension;adrenergic antagonists such as brimonidine or betaxolol, useful as ananti-hypertensives; or any other bioactive agent for which sustained orcontrolled release is medically indicated. The inventive copolymers canbe used in differing types of controlled release formulations.

A flowable delivery system such as in an Atrigel® system, comprising aninventive copolymer, a water-soluble organic solvent such asN-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide,dimethylsulfoxide, polyethylene glycol 200, polyethylene glycol 300, ormethoxypolyethylene glycol 350, and a bioactive agent such asleuprolide, can be advantageously used in a patient to avoid or minimizethe initial burst effect while providing for a prolonged period ofsustained release of the bioactive agent. Likewise, both monolithic andmicroparticulate solid implants incorporating a bioactive agent that arepreformed from an inventive copolymer offer similar benefits of lowinitial burst and prolonged sustained release of the bioactive agent.Other embodiments of sustained release systems and compositions will beapparent to those of skill in the art.

A flowable delivery system such as an Atrigel® system comprising aninventive PLG low-burst copolymer material can be used in the treatmentof a variety of malconditions. The invention provides a method for thetreatment of a malcondition using such a flowable delivery system. Forexample, a flowable delivery system of the invention can be used in thetreatment of prostate cancer with leuprolide, a peptide drug used in thesuppression of testosterone biosynthesis in men, a treatment that isoften medically indicated for patients afflicted with prostate cancer.Implantation of a flowable composition subcutaneously results in theformation of a semi-solid depot as the organic solvent diffused intosurrounding tissues and body fluid, as body fluid diffuses into thebolus. This semi-solid or solid depot then serves to release theleuprolide in a controlled or sustained manner over a prolonged periodof time, which can be in the order of months. Use of the inventivecopolymer materials is effective in reducing the undesirable initialburst effect that can result from the use of art copolymers in a similarsystem.

In a similar manner, other bioactive agents can be used in the treatmentof other types of malconditions when it is medically indicated toprovide the bioactive agent to the patient over the course of weeks ormonths. For example, a flowable delivery system incorporating octreotidecan be used to form a depot for the treatment of acromegaly, thetreatment of diarrhea and flushing episodes associated with carcinoidsyndrome, and treatment of diarrhea in patients with vasoactiveintestinal peptide-secreting tumors.

In the treatment of the malcondition of glaucoma, a flowable deliverysystem of the Atrigel® type incorporating an inventive PLG copolymer andcomprising a bioactive agent suitable for the treatment of glaucoma, forexample a prostaglandin analog such as latanoprost or travoprost ortheir free acid forms, a carbonic anhydrase inhibitor such asdorzolamide or brinzolamide, an a-adrenergic antagonist such asbrimonidine, or a β-adrenergic antagonist such as betaxolol, can beadvantageously used to deliver the bioactive agent over a prolongedperiod while avoid the initial burst effect. The flowable deliverysystem to be used to form a depot either intraocularly, through directinjection into the eyeball, or in proximity to the eye throughimplantation in a nearby tissue.

A flowable delivery system incorporating an inventive PLG low-burstcopolymer and including terbinafine as a bioactive agent can be used fortreatment of onychomycosis of the toenail or fingernail by formation ofa depot underneath the nail.

A flowable delivery system incorporating an inventive PLG low-burstcopolymer and including a steroid can be used to provide a birth controltreatment, wherein a prolonged controlled release is desired to controlunwanted fertility and to suppress ovulation while avoiding potentialside-effects from steroid overdose that could occur due to initial burstwhen using an art copolymer for this application.

A flowable delivery system incorporating an inventive PLG low-burstcopolymer and including an antibiotic, e.g., dapsone, can be used in thetreatment of chronic infection.

A flowable delivery system incorporating an inventive PLG low-burstcopolymer and including an antipsychotic, e.g., risperidone, can be usedin the treatment of psychosis.

A flowable delivery system incorporating an inventive PLG low-burstcopolymer and including rapamycin, an immunosuppressant, can be used inthe treatment of cancer or in controlling tissue rejection as in tissueor organ transplantation.

A flowable delivery system incorporating an inventive PLG low-burstcopolymer and including an antiviral agent, e.g., AZT, can be used inthe treatment of a viral infection.

Other conditions and appropriate medicaments for their treatment will beapparent to those of skill in the art.

EXAMPLES

Certain examples are provided below in order to assist in understandingembodiments of the present invention; they should not, however, beconsidered as limiting the present invention, which are described in theclaims.

Introduction to the Purification of Biodegradable Polymers to Improve InVivo Release Kinetics.

The release of many active agents such as peptides and proteins from theAtrigel® system can occur at a higher than optimal rate during the first24 hours after implantation under certain conditions. The polymers ofthe instant invention in combination with the Atrigel® systems resultsin a unique combination that provides substantially improved low-burstrelease rates.

The polymer used in the Atrigel® system is purified by dissolution in asolvent and precipitation in a non-solvent, then dried. The solvent andnon-solvent can be miscible.

Specifically, the polymer can be dissolved in dichloromethane andprecipitated into methanol. As described below, the formulationcontaining purified material was compared to the same formulationcontaining a polymer that was not purified by the inventive method. Theformulation containing purified material displayed a lower drug releasein the first 24 hours and later time points. Use of an inventivecopolymer in the Atrigel® system is thus shown to improve in vivo drugrelease kinetics, especially with respect to drug release during thefirst 24 hours after administration.

Example 1 Purification of Acid Terminated 85/15poly(DL-lactide-co-glycolide) (85/15 PLGH)

Test articles were prepared with and without the purified copolymer andcompared with the starting copolymer (the copolymer prior to carryingout steps of dissolution and precipitation) in a 24 hour release study.The starting copolymer in this Example was an acid-terminated form ofpoly(DL-lactide-co-glycolide), meaning that one end of the molecularchains making up the copolymer material bear a carboxylic acid group.Forty-eight grams of 85/15 PLGH with an inherent viscosity of 0.25 dL/gwas dissolved in 100 mL of dichloromethane. The polymer solution waspoured into a 2L beaker containing 500 mL of methanol with vigorousstirring. The precipitated polymer formed a soft mass. Thedichloromethane-methanol solution was decanted and 200 mL of methanolwas added for 5-15 minutes to further extract the dichloromethane. Themethanol was decanted from the container and replaced with 100 mL ofmethanol for an additional 5-15 minutes to further extract thedichloromethane.

The polymer mass was removed from the container and placed in aTeflon-lined glass dish and dried under vacuum at 40° C. for 48 hours.The dried polymer was removed from the vacuum oven, ground into a powderand dried another 24 hours at 40° C.

Preparation of Atrigel® Formulations

Solutions (45% w/w) of both purified and unpurified copolymers inN-methylpyrrolidone (NMP) were prepared. Stock solutions were preparedby weighing a known amount of each copolymer into individual 20 mLscintillation vials. The appropriate amount of NMP was added to eachpolymer and the mixture placed in a jar mill. The vials were mixed atleast overnight, producing a visually clear polymer solution. Thepolymer solutions were gamma-irradiated. The characterization data forthe purified and unpurified polymer in solution is shown in Table 1.

TABLE 1 Characterization Data for Purified and Unpurified Polymer;Molecular weight and Polydispersity Data is for Post Gamma-IrradiatedPolymer Mole % Mole % DL-lactide Glycolide Wt % DL- Wt % MW PDI in inlactide Glycolide Sample (kDa) [1] Polymer Polymer Monomer MonomerPurified 19 1.6 84.7 15.3 0.5 0.1 Polymer Unpurified 18 1.7 84.5 15.52.3 0.1 Polymer [1] Polydispersity Index = Weight Average MolecularWeight/Number Average Molecular Weight

An octreotide acetate-citric acid mixture was prepared by dissolving 4 gof octreotide acetate, and 0.7550 g citric acid (1:1 mole:mole) into 30mL HPLC grade water. The solution was stirred until all of the solidswere in solution. The solution was divided into 5 separate vials, andwas frozen at −86° C. for 1 hour. The vials were then lyophilized for 2days.

A stock solution of drug was prepared by dissolving 1.35 g of drug in4.65 g HPLC grade water, yielding a 22.5% (w/w) stock solution. Drugcontaining syringes (“B” or male syringes) were prepared by pipetting500 mg of octreotide stock solution into 1.25 mL BD syringes, followedby lyophilization for 24 hours. Polymer solution containing syringes(“A” or female syringes) were prepared by weighing 637.5 mg polymersolution into 1 mL female syringes.

Prior to administration in rats, the two syringes were mated togetherand the contents mixed by forcing the contents of the syringes betweenchambers a predetermined number of times. Once the contents of the twosyringes were mixed, a 19 gauge thin-wall needle was attached to thefemale syringe and approximately 100 mg of Atrigel® formulation injectedsubcutaneously into a rat.

At predetermined times, five rats per group were euthanized with carbondioxide and the Atrigel® implants recovered. The implants were analyzedfor the amount of drug remaining in the implant by HPLC and the % drugrelease was calculated.

The drug release from the purified polymer test article was compared tothe test article with the same polymer without purification. The data,means and standard deviations are displayed in Table 2 and in FIG. 1.

TABLE 2 Mean % Drug Released After 24 Hours Purified Control PurifiedControl Polymer Polymer Polymer Day 1 Polymer Day 1 Day 21 Day 21 %Release % Release % Release % Release 11.64 20.70 43.3 51.6 13.00 28.0042 63.5 8.60 16.20 50.6 52.7 10.53 15.40 42.2 50.5 8.70 15.70 35.8 49.7Mean 10.49 19.20 42.78 53.60 Std. Dev. 1.90 5.37 5.27 5.65

Example 2

In Example 2, test depots comprising octreotide as a bioactive agentwere prepared from polymers purified by two different methods andcompared to a test article prepared from the same polymer in unpurifiedform, in a 28 day controlled release study in rats.

Example 2a Purification of Polymer in NMP/Water/Ethanol (Control Method)

A purification technique was developed that involved dissolving thepolymer in N-methyl pyrrolidone (NMP) and precipitating the polymersolution into a water/ethanol solution. NMP, water, and ethanol can haveadvantageous properties when used in pharmaceutical preparationscompared to dichloromethane (methylene chloride) and methanol.

The polymer was dissolved in NMP for use in the delivery system. Onehundred grams (100 g) of 85/15 PLGH was added to 400 g of NMP in a 2 LNalgene bottle. The bottle was shaken to disperse the polymer and placedon a roll mill overnight to dissolve the polymer.

A 9.5 L container was equipped with an overhead stirrer set off-centerand filled with 4 L of water. With the overhead stirrer at approximately1250 rpm (Setting 3), the polymer solution was slowly added to thecontainer through a funnel over a 5 minute time period. The resultingpolymer suspension was stirred for 30 minutes at 1250 rpm. The stirringwas then slowed to about 500 rpm while 3 L of water and 1 L of ethanolwas added to the container. The polymer suspension aggregated and wasredispersed by increasing the stir speed to approximately 800 rpm(setting 2.5) and manually breaking up the aggregate.

After 30 minutes the stirring was stopped and the suspension allowed tosettle and separate for 20 minutes. A small amount of solids rose to thesurface, but the majority of material settled to the bottom of thecontainer. Four liters (4 L) of solvent were decanted from the containerand stirring was resumed approximately 800 rpm while 3 L of water and 1L of ethanol was added. Stirring was continued for 30 minutes and thenthe suspension was allowed to settle and separate for 30 minutes. Fourliters (4 L) of solvent was then decanted from the container.

Stirring was resumed at the 800 rpm setting and 3 L of water and 1 L ofethanol again added and stirring continued for 2 hours. The mixture wasallowed to settle for 15 minutes. Four liters (4 L) of water was addedto the container and stirred for an additional 1-2 hours. The suspensionwas filtered and the filter cake was spread into a Teflon lined Pyrexdish and dried in a vacuum oven at room temperature for approximately 70hours. The weight was recorded and placed back in the vacuum oven anddried under vacuum at 30-40° C. for an additional 19 hours. The driedpowder was transferred to a glass jar.

Example 2b Purification of Polymer in Dichloromethane/Methanol (Testmethod)

One hundred grams (100 g) of 85/15 PLGH was added to 393 g ofdichloromethane (DCM) in a 1 L Nalgene bottle. The bottle was shaken todisperse the polymer and placed on a roll mill overnight to dissolve thepolymer.

A 9.5 L container was filled with 4 L of methanol. The polymer solutionwas slowly added to the methanol through a funnel in a thin streamwithout stirring. The polymer formed a soft mass in the bottom of thecontainer. The material was manipulated with a stirring rod to exposefresh surface area to assist in DCM diffusion into the methanol.

After 15 minutes, the solution was decanted and 2 L of fresh methanolwas added. The material was again manipulated to generate new surfacearea to allow the DCM to diffuse out of the polymer and into themethanol. The soft mass was periodically kneaded to press out solventand force DCM into the methanol.

After about 5 hours the excess solvent was pressed out of the softpolymer mass and it was placed in a Teflon lined Pyrex dish, placed in avacuum oven and solvent removed by vacuum at room temperature. Afterabout 24 hours, the brittle material was ground to a powder and placedback in the vacuum oven to further dry. After 48 hours, the polymer wasweighed and placed in the vacuum oven at 30-40° C. The polymer wasweighed again after 19 hours and the weight had not changedsignificantly. The polymer was placed in Nalgene bottle. The final yieldwas 61 g.

Example 2c Preparation of Bulk Atrigel® Formulations

Fifty percent (50%) polymer solutions in NMP were prepared by weighingboth components into a 20 mL glass vial. The vial was placed on a rollmill to dissolve the polymer into the NMP. The following copolymersamples were prepared:

-   Polymer 2A: 5 g of (85/15 PLGH purified in NMP/Water/Ethanol) was    dissolved in 5 g of NMP.-   Polymer 2B: 5 g of (85/15 PLGH purified in DCM/methanol) was    dissolved in 5 g of NMP.-   Polymer 2C: 5 g of (85/15 PLGH unpurified) was dissolved in 5 g of    NMP.

The bulk solutions were irradiated and filled into syringes. The bulkformulations were characterized by gel permeation chromatography tomeasure the molecular weight of the polymer in the solution afterirradiation. Molecular weight data are given in Table 3.

TABLE 3 Characterization Data for Purified Polymers and Control Polymerafter Gamma Irradiation Purification Molecular Weight Polymer DispersityPolymer Technique (kDa) (n = 2) Index (n = 2) 2A NMP/Water/EtOH 21 1.82B DCM/MeOH 21 1.7 2C Unpurified 21 1.7

Example 2d Preparation of Drug Loaded Syringes

OTCA (Octreotide acetate (2.33 g) and citric acid (0.43 g)) weredissolved in 21.24 g of water. The appropriate amount of solution wasweighed into 5 CC syringes and frozen at −80° C. and lyophilized.

Example 2e Preparation of Test Depots

Prior to administration in rats, the syringe containing the Atrigel®formulation was mated to the syringe containing the lyophilized drug andthe contents were mixed by forcing the contents of the syringes betweenchambers a predetermined number of times. Once the contents of the twosyringes were mixed, a 19 gauge thin-wall needle was attached to thefemale syringe and approximately 100 mg of Test Article injectedsubcutaneously into a rat.

750 mg of constituted product was prepared by mixing 112.5 mg drug in637.5 mg of ATRIGEL® vehicle. The homogeneous mixture was then weighedinto a 1.5 ml, syringe and a 19 gauge thin-wall needle was attached.Each rat receive approximately 100 mg of the constituted product viasubcutaneous injection.

At predetermined times five rats per group were euthanized with carbondioxide and the Atrigel® implants recovered. The implants were analyzedfor the amount of drug remaining in the implant by HPLC and thecumulative percentage of drug released calculated. The data for the 1-,14-, and 28-day time points are shown in Table 4 and FIG. 2.

TABLE 4 Release Data Day 1 Day 14 Day 28 Avg. Wt. % Day 1 Avg. Wt. % Day14 Avg. Wt. % Day 28 OTC Avg. Wt. % OTC Avg. Wt. % OTC Avg. Wt. % n = 5Released STD Dev Released STD Dev Released STD Dev NMP 12.83 2.79 43.738.34 54.45 3.94 (Atrix) CH₂Cl₂ 14.75 9.22 34.09 7.16 45.91 3.25 (Atrix)Not 19.16 4.94 44.46 10.28 48.93 3.57 Purified

The test articles prepared with both forms of purified polymers hadinitial lower burst that the test article using the unpurified polymer,the percent release of octreotide from the NMP and DCM copolymerpreparations at Day 1 being the same within experimental error butsignificantly lower than from the unpurified polymer. However, at Days14 and 28, the test depot containing a polymer purified by the DCMmethod indicated a significantly slower release of octreotide than didthe depots formed from the NMP-purified polymer or from the unpurifiedpolymer.

The molecular weight data in Tables 1 and 3 illustrate that thepurification did not significantly alter the weight average molecularweights of the polymers in the gamma irradiated formulations. Thepurification did, however, narrow the molecular weight distribution orpolydispersity index when compared to the control.

To further understand this phenomenon, an 85/15 PLGH polymer purchasedfrom an outside vendor (referred to as unpurified polymer 2D) waspurified in DCM and methanol using a method similar to that describedabove. This lot of purified polymer was labeled polymer 2E. The polymerswere characterized by GPC and NMR. In addition, the impurities thatremained in the DCM/methanol solvent were collected and characterized byGPC and NMR. The data appear in Table 5.

TABLE 5 Characterization Data for Raw and Purified 85/15 PLGH andResidue of Purification Mole % of Monomer in Polymer Weight % Monomer MWDL- DL- Sample (kDa) PDI [1] lactide Glycolide lactide GlycolideUnpurified 23 1.73 84 16 1.7 0.1 polymer 2D Purified 23 1.66 83 17 0.5 0Polymer 2E Polymer 4 1.5 83 17 1.4 0 Residue 2E [1] PolydispersityDispersity Index = Weight Average Molecular Weight/Number AverageMolecular Weight

The NMR data indicated that the purification removed residual monomer.The weight percent lactide was reduced from 1.7 wt % to 0.5 wt % and theresidual glycolide monomer was reduced from 0.1 wt % to 0 wt %. The GPCdata again shows that while the weight average molecular weight did notsignificantly change, the polymer dispersity index (PDI) decrease from1.73 to 1.66, indicating removal of a low molecular fraction of thepolymer. This low molecular weight fraction left in the solvent mixturehad an average molecular weight of only 4 kDa.

Example 2f Method of Analysis of Residual Octreotide in RecoveredImplants I. Implant Preparation

-   A. Implants are received from the Pre-clinical department in labeled    20 ml Scintillation vials-   B. The implants are frozen at −86° C. for at least 1 hour-   C. The implants are then lyophilized for 4 or more hours or until    dry-   D. Dried implants are then minced with scissors

II. Implant Extraction

-   A. An extraction solvent solution consisting of 70:30 DMSO:MeOH+1%    PEI is prepared by measuring 700 ml of DMSO and 300 ml of MeOH in a    graduated cylinder. The solvents are added to a 100 ml bottle. The    bottle is shaken to mix the solvents. 10 g of PEI is weighed into a    250 ml beaker. The PEI is transferred to the solution by adding    small amounts of the mixed solution to the PEI beaker and swirling,    then adding the solution back to the solvent bottle. The process is    continued until no PEI remains in the beaker.-   B. 5.0 ml of extraction solvent is added to the minced implants    using a micro-pipetter-   C. The implant solution is placed in a horizontal shaker set of 37°    C., 200 RPM. The samples are shaken overnight.

III. Extraction Solution Filtration and Dilution

-   A. 2 ml of implant extract is filtered using a 3 ml syringe and 0.2    um nylon syringe filter into a 10 ml test tube. 1 ml of the filtrate    is aliquoted to second tube using a 1 ml micropipetter.-   B. A dilution solvent consisting of 50:50 Acetonitrile: water is    prepared by measuring 500 ml of Acetonitrile (ACN) in a graduated    cylinder and adding the solvent to a 1000 ml bottle. 500 ml of water    is added to the bottle and the bottle is shaken to mix the solvents.-   C. 4.0 ml of dilution solvent is added to the aliquoted implant    extract. The solution is vortex mixed until no phase separation is    observed.-   D. The diluted extract is filtered using a second 3 ml syringe and    nylon syringe filter. The extract is filtered into a 2 ml HPLC vial    and capped for HPLC analysis.

IV. HPLC Analysis A. Standard Curve Preparation

A standard curve is prepared using the Octreotide drug powder (OTCA orODP) used in the test articles for the study the implants are collectedfrom. 10.00 mg of Octreotide drug powder is weighed into a 10 mlvolumetric flask. The flask is brought to volume using Octreotide mobilephase (see section IV B, mobile phase preparation.). The flask is vortexmixed until all powder is in solution. The prepared stock solution isfurther diluted with mobile phase to prepare a standard curve using 1000ml and 100 ml micropipetters. The dilutions are made into 2 ml HPLCvials. The dilution volumes are outlined below.

B. Mobile Phase Preparation

2 L of 65:35 PO4:ACN buffer (octreotide mobile phase) is prepared byweighing 14.047 g of Na₂HPO₄*7H₂O into a weigh boat. The powder is addedto a 2 L volumetric cylinder. 0.7839 g of NaH₂PO₄ is weighed into aweigh boat and added to the cylinder. HPLC grade H₂O is added to thecylinder to the 1300 ml mark. A stir bar is added and the solution isstirred until all solids are dissolved. The pH of the buffer is adjustedto pH 7.4 using ortho phosphoric acid. Acetonitrile is the added to theflask to the 2000 ml mark. The mobile phase is stirred well ad degassedfor 10 min using a sonicator bath.

C. HPLC Parameters:

-   The analytical column used is a Merck LiChroSphere 125×4 mm RP    select B 5 um. After each run, two cleaning steps are run. The first    is a 30 min run with 70:30 H₂O:ACN, the second is a 30 min run with    30:70 H₂O:ACN.

TABLE 6 Release of a peptide from an ATRIGEL ® delivery system*. 24 Hour% Release Average (Standard Deviation) Delivery System PurifiedUnpurified Study Active Solvent Polymer Polymer Polymer ATRS 963 0.16%PYY 55% NMP 45% 50/50 45.6 (9.9) 57.6 (7.7) PLGH (IV 0.37) QRS- 0.8% 60%NMP 40% 65/35 23.9 (6.8) 56.5 (7.2) R041-05 Leuprolide PLGH Acetate(0.37 IV) QRS- 10% 50% NMP 50% 85/15 14.2 (8.6) 27.9 (7.5) R026-05GHRP-1 PLGH (plus citric (25 kDa) acid) ATRS-606 10% 50% NMP 75/25  5.3(1.7) 22.4 (3.0) and QRS- GHRP-1 PLGH (IV R026-05 (plus citric 0.24)acid) *copolymer purification was with dichloromethane/methanol.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements will be apparent to thoseskilled in the art without departing from the spirit and scope of theclaims.

1-12. (canceled)
 13. A controlled release formulation comprising aflowable delivery system comprising a biocompatible, biodegradable,non-hydrolyzed PLG low-burst copolymer material for a controlled releaseformulation having a weight average molecular weight of about 10kilodaltons to about 50 kilodaltons and a polydispersity index of about1.4-2.0 and from which a removed copolymer fraction characterized by aweight average molecular weight of about 4 kDa to about 10 kDa and apolydispersity index of about 1.4 to 2.5 has been separated, an organicsolvent, and leuprolide. 14-23. (canceled)
 24. A method of treatment ofa condition in a patient, comprising providing the patient with atherapeutically effective amount of the flowable delivery system ofclaim 13 comprising a medically indicated bioactive agent, wherein thecondition is prostate cancer and the bioactive agent comprisesleuprolide. 25-40. (canceled)